Abstract
The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.
Highlights
The fermentation of 13C-labeledethanol and acetate make ATP results in theproduction of acetate, a required into butyrate and caproate by Clostridium Muyveri carbon source, from ethanol and in theproduction of excess has been studied byusing 13C NMR
When C.kluyveri cells were incubated in buffer containing [l-13C]ethanol,but no acetate, the 13CNMR spectrum showed rapid but limited conversion of ethanol to caproate as evidenced by the appearance of the caproate C1,C3, and C5 resonances at 184.2,25.7, and 21.9 ppm, respectively
Upon addition of unlabeled acetate, the I3Cresonance of the acetate C1 appearedand the resonance corresponding to Portions of this paper are presented in miniprint at the end of this paper
Summary
From the $Department of Food Science and Technology, University of California, Davis, California 95616 and the $Gates and CrettirzLaboratories, California Znstitute of Technology, Pasadena, California 91125. ATP is produced from acetyl-coA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. The most likely mechanism was thought to bethrough the use of an electron-transport system involving CoA-linked metabolites such as crotonyl-CoA [2] It has since been demonstrated by the measurement of growth yields rapid initial production of acetate indicates that the organism is capable of surviving on the amount of rapid production of ATP. The organism generates ATP from acetyl-coA via the phosphotransacetylase and acetate kinase reactions which form free acetate asa co-product.
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